Method and apparatus for detecting and classifying nucleic acid particles

ABSTRACT

Free floating viruses are detected and sized by a method which combines fluorescent staining with the observation of a modulation of the fluorescence intensity by Brownian motion of the particles in combination with a spatial filter. Intensity modulation and fluorescence data provides a set of descriptors useful in distinguishing the type of virus involved, particularly if also combined with data regarding the scattering of light by the particles. The method and apparatus are also usable in connection with detection and classification of other biological particles having the same order of magnitude of size as viruses.

United States Patent 91 Hirschfeld Mar. 18, 1975 METHOD AND APPARATUS FOR DETECTING AND CLASSIFYING NUCLEIC ACID PARTICLES [75] Inventor: Tomas Hirschfeld, Framingham,

Mass.

[73] Assignee: Block Engineering, Inc., Cambridge,

Mass.

[22] Filed: July 2, 1973 211 Appl. No.: 375,807

[52] US. Cl. 250/458, 250/461 [51] Int. Cl. G01t 1/16 [58] Field of Search 250/302, 304, 361, 458,

[56] References Cited UNITED STATES PATENTS 3,497,690 2/1970 Wheeless, Jr. et a] 250/304 3,657,537 4/l972 Wheeless, Jr. et al. 250/304 Primary Examiner-Archie R. Borchelt Assistant Examiner-Davis L. Willis Attorney, Agent, t)! FirmSchiller & Pandiscio [57] ABSTRACT Free floating viruses are detected and sized by a method which combines fluorescent staining with the observation of a modulation of the fluorescence intensity by Brownian motion of the particles in combination with a spatial filter. Intensity modulation and fluorescence data provides a set of descriptors useful in distinguishing the type of virus involved, particularly if also combined with data regarding the scattering of light by the particles. The method and apparatus are also usable in connection with detection and classification of other biological particles having the same order of magnitude of size as viruses.

25 Claims, 6 Drawing Figures l l l l [FILTE LlGHT SOURCE l FREQUENCY 'ANALYZERS Y I DETECTORS R FILTER l sum 1 OF 2 FREQUENCY ANALYZERS I 30 28 26, f f f DETECTORS I l r -32 n/1X55 FILTER F| LTER BEAM SPLITTER VIEWING oPTIcs.

STAINED SAMPLE PROJECTION OPTICS KIB APERTuRE FILTER z F /G.

LIGHT SOURCE METHOD AND APPARATUS FOR DETECTING AND CLASSIFYING NUCLEIC ACID PARTICLES The present invention relates to clinical laboratory methods and apparatus and more particularly to .detection and classification of virus particles.

Virus particles, responsible for a wide variety of plant and animal pathology, comprise exceedingly small particles sized on the order of a wavelength of visible light or smaller (3,500 to 200 Angstroms) and consist essentially of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) surrounded by a protein shell. The ratio of nucleic acid content to protein content varies from 1:100 to 1:2.

Viruses are usually studied in animal, bacterial or plant hosts or in cell cultures derived from tissues of the hosts. Virus presence is inferred by observing the development of' infectious symptoms in a patient due to viral infection. The long time involved and the danger of spread of the infection limit the desirability of this technique.

Virus-suspect specimens may be subjected to a variety of chemical reactions using known techniques of immunochemistry to detect and classify viral species, if any, therein. The reactions involved are highly specific and a large number of specific antibodies are necessarily involved. At present however, only a limited number of such antibodies are known. Culturing suspect specimens and examination in the electron microscope to observe differences is limited in its effectiveness by the long time involved in culturing and sample preparation. It has also been known in the prior art to produce a suspension of a suspect specimen in liquid, illuminate the suspension with coherent light and observe backscattered light by heterodyne spectometry to obtain data on Brownian motion of suspended particles correlatable with size of the particles. In other words, the method measures particle velocity by observation of the Doppler shift. The use of coherent and essentially monochromatic radiation permits measurement even of the comparatively slow Brownian velocities. However, inadequate ability to descriminate among viral species and between viral particles and similarly sized cell debris in the specimen limit the utility of this technique.

It is therefore an important object of the present invention to provide method and/or apparatus for virus detection avoiding the above cited problems of the prior art.

It is a further object of the invention to provide classification among viral species present in a suspectsample consistent with the preceding object.

It is a further object of the invention to provide a short time of speciment treatment and data extraction consistent with one or more of the preceding objects.

It is a further object of the invention to provide inexpensive and simple apparatus and/or operating steps consistent with one or more of the preceding objects.

According to the invention, virus-suspect specimens are treated by the following process:

a. Taking from the patient a body fluid sample containing a suspension to be examined for virus, and treating the sample with one or more fluorescent dyes specific to a nucleic acid;

b. Illuminating the sample with light in an absorption band of the dye or dyes and observing fluorescence, if any, from the stained sample at spectral peaks associated with deoxyribonucleic or ribonucleic acid, and substantially simultaneously spatially filtering either or both the excitation illumination and the emitted fluorescence, whereby the 5 observed fluorescent intensities of dyed particles is modulated by the Brownian motion of such dyed particles. Scattering behavior and/or polarized light response may also be observed.

' Spatial filtering of observed fluorescence intensities is achieved in the present invention in one or two basic ways. In the first method, the radiation used to illuminate the particles is spatially filtered: For example, a regular grid or a simple aperture is placed between the illumination source or optics and the suspension of particles, thereby providing one or more zones of high and low illumination. ln the second method the fluorescence radiation itself is spatially filtered, for example, by placing a grid between the suspension of particles and the viewing optics, thereby providing zones within which fluorescence can or cannot be observed. In either case, as the particles move from zone to zone by Brownian motion, the fluorescent emission will be modulated to produce a repetition rate spectrum which is a function of the velocities of the fluorescing particles. The particle velocities in turn are a function of the particle masses.

By wavelength filtering, one can readily. identify the wavelength of the fluorescent emission and thereby determine the particular nucleic acid to which the fluorochrome is bound. This, together with simultaneous observation of the intensity modulation of the fluorescing particles serves to distinguish viral-like particles from background and provides-useful viral classification def scriptors. The number of useful descriptors can be enhanced through correlation of simultaneous observations of two-color fluorescent modulation of intensity by Brownian motion and further enhanced by correlation of polarized light response and scattering data with the measurements of intensity modulation by Brownian motion. The method and apparatus of the invention are also usable in connection with detection and classification of other biological particles having the same order of magnitude of size as viruses, i.e., submicron size.

A conventional laboratory microscope may be modified by the addition of aperture, filters and afluid-well specimen holder in accordance with the present invention to carry out the above described process. Preferably, the microscope is provided with multiple detector channels for simultaneous readout of intensity modulated wavelengths corresponding to each of DNA and RNA. However, a single channel may also be utilized with changes of filters for sequential observation for the two colors to be detected. Scattering data is taken separately or together with the fluorescence data. Readout is preferably done automatically, by photodetectors coupled to frequency analyzers for each readout channel. The frequency analysis output for each channel is a graphical or digital representation of intensities of signal at various frequencies. Whether a signal is generated at all is determined by coincidence of the color selective properties of the detection system with the fluorescent emission wavelength of the fluorochrome attached to the viral particle, which is dependent on the selection of fluorochrome and whether it is linked to a DNA or RNA-containing particle.

These and other objects, features and advantages of the invention will be apparent from the following detailed description with reference therein to the accompanying drawing in which:

FIG. 1 is a block diagram of apparatus for detection of viral species and classification by composition of a particular nucleic acid; and

FIGS. 26 are graphical representations of the relationship of light intensity to intensity modulation frequency in operation of the invention, all graphs having the same coordinate increments in abscissa and ordinate.

According to a preferred embodiment of the invention the known techniques of tissue culture preparation are used to obtain a virus-suspect sample. The known techniques are modified to the extent of being carried out in a time tooshort to produce the growth areas of cell destruction associated with plaque preparation. The sample so obtained is suspended in a liquid. Alternatively, a natural body fluid suspension of virus particles [e.g., plasma or spinal fluid] may be directly utilized. The suspension is mixed with fluorochrome dye stains which are soluble in the fluid and specific to the viral or other small nucleic-acid-containing particle to be detected.

The apparatus of a preferred embodiment of the invention comprises a modified microscope with a laser or other high intensity light source 12 for projecting light through a wavelength filter 14, a spatial filter 18, and conventional projection optics 16. Filter 14 restricts the illumination to an excitation band of the fluorochrome dye. Spatial filter 18 may be a single aperture or a multiaperture filter such as a grid or the like. The spatial filter is imaged in the plane of the sample which is held in a transparent fluid well in sample holder slide 20. It will be understood the spatial filter may precede the slide or the slide may precede the spatial filter in the light path. In the latter case, the sample is imaged onto the spatial filter by viewing optics 22.

Spatial filters are well known to those skilled in the optical arts and may take the form of gratings, grids, annuli, and the like. Each defines one or more zones or edges between a relatively light-transmitting element and a relatively nontransmitting element. It will be apparent that as a particle, moved by Brownian forces, crosses the edge or zone of a filter to a lighttransmitting area, the detector will see that particle somewhat as a light burst or scintillation. As the particle crosses the edge into a non-transmitting area, the

burst is extinguished. Thus, assuming that a particle travels in a straight line across a grid, the observed intensity of emission from the particle will fluctuate between maximum and minimum values at a frequency depending on the grid spacing and the particle velocity. Theoretically, for optimum modulation and duty cycle, the grid spacing should, at least in order of magnitude, match the particle size. For particles of submicron size, such matching is a difficult task. However, spatial filters approaching diffraction-limited apertures for the wavelengths employed, although having aperture sizes far greater than viral dimensions, will nevertheless provide quite useful results. e

The light passing through the sample and/or emitted by the sample is viewed through conventional viewing optics 22, a beam splitter 24, and photoelectric detectors 26, 28, 30. Detectors 26 and 28 may have wavelength filters 32 and 34 associated therewith to limit their-effective spectral rangesv to different fluorescence bands or may themselves have limited spectral ranges. Detector 30 may also be provided with a wavelength filter and known means 48 to mask out direct radiation from light source 12 so that detector 30 sees only scattered light.

The electrical output signals from the detectors are amplified by preamplifiers 36, 38 and 40 and applied to known frequency analyzers 42, 44, 46 which are synchronized by mechanical or electronic means such as a common clock circuit associated therewith. The outputs of the frequency analyzers are applied to data reduction apparatus which may be a strip chart recorder and/or a computer.

To obtain desired specificity of nucleic acid staining, solubility, and the property of fluorescence at desired wavelengths under excitation by light of desired wavelengths, one may use the cationic dyes: acridine orange or yellow GR, quinacrine mustard, ethydium bromide, pyronine B, aurophosphine, euchrysine 2GNX and 3R, vesuvin, rhodamine S, B and 6G, coriphosphine O, civanol, acriflavine, atabrine, phosphine, benzoflavine, rheonine A, thioflavine T and berberine. Many other dyes can be used with appropriate adaptation of sample chemistry or the light source to suit such dyes.

With an appropriate spatial filter, the apparatus of FIG. 1 can be operated with a 0.05 cc. sample having a particle concentration of about 10 particles (of approximately 200 to 3,500 Angstrom size) per cubic mm., to make classification measurements in one minute or less. Sample handling can be accomplished at a rate of 5 minutes or less per sample. Accuracy of siz classification can be with t 3%.

The outputs of the frequency analyzer for a single sample are shown schematically in FIGS. 2-5 which are intensity vs. modulation frequency graphs (in Hertz). The sample was studied with acridine orange and its fluorescence was measured through different channels or in a single channel with changes of detection filtering between observations.

FIG. 2 is a graph taken with a filter passing an appropriate wavelength (5,500 A in this example) to detect the presence of DNA particles. The location of each of peaks A, B, C on the resulting curve are proportional to the equivalent diffusional diameters of various DNA- containing particles. Peak heights and areas are proportional to quantities of the DNA-containing particles of the sizes indicated by peak location.

The curve in the graph of FIG. 3 is created by the frequency analyzer and its printout device when using a wavelength filter (5,500 A in this case) chosen to detect RNA-containing particles.

Clearly peak B of the curve in the graph of FIG. 2 corresponds to a DNA-containing virus because there is no corresponding peak in FIG. 3. Similarly peak D of FIG. 3 corresponds to an RNA-containing virus. As viruses have to belong in one of these types, the continued presence of peaks A and C indicates a slightly shifted DNA spectral behavior which indicates a structural change in the DNAs arrangement. These flucturations in the overlap between both channels have recognition value.

FIG. 4 shows the result of a repeat run with polarized filter specific for scattering due to the total particle mass. The illuminating source is an argon laser. This filter is chosen to correspond to the illumination, and is 4,880 Angstroms in this case. I

It is clear from comparison of FIG. 4 with FIGS. 2 and 3 that the peaks E, F, G, H, and I of FIG. 4 are due to the presence of particles that do not contain nucleic acid and may be disregarded.

FIG. 5 shows the result of a repeat run with the 4,880 Angstrom polarized filter with a different orientation.

The ratio between the readings of FIGS. 4 and 5 describes the polarization in the particle scattering and thus the particles elongation.

The average of the curves of FIGS. 4 and 5 gives the total particle mass, whose ratio to the FIGS. 2 or 3 curves gives the nucleic acid/total weight ratio in the particle.

FIG. 6 is a similar plot, for apparatus calibration purposes, of intensity vs. modulation frequency, using the apparatus of FIG. 1 with a single tiny aperture as a spatial filter to detect suspended particles of 880 Angstrom polystyrene spheres, a known phage virus, and 2,340 Angstrom polystyrene spheres. The polystyrene spheres were observed in scattered light.

The results are shown, respectively, on the curves marked 880, V, and 2,340. The half-width of the halfheights of the respective curves occur at 34.5, 13.2 and 27.5 Hertz indicate the frequencies relatable to known diameters of the particles.

It is evident that those skilled in the art, once given the benefit of the foregoing disclosure, may now make numerous other uses and modifications of, and departures from the specific embodiments described herein without departing from the inventive concepts. Consequently, the invention is to be construed as embracing each and every novel feature and novel combination of features present in, or possessed by, the apparatus and techniques herein disclosed and limited solely by the scope and spirit of the appended claims.

What is claimed is:

1. Apparatus for detecting and classifying, in a fluid specimen, submicron-dimensioned particles containing a nucleic acid and stained with a fluorescent dye specific to said nucleic acid, said apparatus comprising,

a radiation source for providing illumination within an excitation band of said dye,

means for directing said illumination into said specimen, means for viewing fluorescence emission produced from said dye responsively to said illumination, and

means for spatially filtering at least one of said illumination and said fluorescence emission so as to provide intensity modulation of said emission due to Brownian motion of said particles.

2. Apparatus as defined in claim 1 wherein said means for spatial filtering is disposed in the optical path between said source and said specimen.

3. Apparatus as defined in claim 1 wherein said means for spatial filtering is disposed in the optical path between said means for viewing, and said specimen.

4. Apparatus as defined in claim 1 wherein said means for spatial filtering includes one or more light transmissive portions, substantially all of which are dimensioned to be as closely matched to the average dimension of said particles as the wavelengths of said illumination permits.

5. Apparatus as defined in claim 1 including means for detecting intensity modulation of fluorescence produced by Brownian motion of said particles in combination with said means for spatially filtering.

6. Apparatus asdefinedin claim 5 including means for frequency analyzing said intensity modulation.

7. Apparatus as defined in claim 5 including means for dividing said fluorescence emission into a plurality of beams, said means for detecting including a plurality of wavelength filters each .disposed in the path of a respective one of said beams and a corresponding photospecimen, of submicron dimensioned particles which contain a nucleic acid, which particles are stained by a fluorescent stain specific to said nucleic acid, said process comprising the steps of illuminating said fluid with radiation at an excitation wavelength of said stain observing substantially any fluorescent emission produced from said stain responsively to the excitation illumination; and spatially filtering at least one of the excitation illumination and said fluorescent emission so that the observed fluorescent emission is modulated as a function of the Brownian velocity of the stained particles. 12. Process as'defined in claim 11 including the step of first staining said specimen with said fluorescent stain.

13. Process as defined in claim 11 including the steps of suspending the stained particles in a fluid to form said fluid specimen.

14. Process as defined in claim 11 wherein said stain is specific to DNA.

15. Process as defined in claim 11 wherein said stain is specific to RNA.

16. Process as defined in claim 11 including the steps of staining said particles with both DNA-specific and RNA-specific fluorochromes, and

filtering the fluorescence emission at separate wavelengths corresponding to fluorescent wavelengths of stains produced by linkage of said fluorochromes to DNA and RNA, respectively.

17. Process as defined in claim 11 and further comprising the steps of viewing light scattered from said particles and correlating the observation thereof with the observed spatially filtered fluorescent emissions to determine the ratio of nucleic acid mass to total mass of suspended particles.

18. Process as defined in claim 17 wherein the fluorescent emissions and scattered light are viewed simultaneously.

19. Process as defined in claim 11 wherein only said excitation illumination is spatially filtered.

20. Process as defined in claim 11 wherein only said observed fluorescent emission is spatially filtered.

DNA and RNA.

24. Apparatus as defined in claim 10 wherein said means for detecting and measuring scattering is sensitive to polarization of the scattered illumination.

-25. Process as defined in claim 17 including the step of measuring polarization of the light scattered from said particles. 

1. APPARATUS FOR DETECTING AND CLASSIFYING, IN FLUID SPECIMEN, SUBMICRON-DIMENSIONED PARTICLES CONTAINING A NUCLEIC ACID AND STAINED WITH A FLUORESCENT DYE SPECIFIC TO SAID NUCLEIC ACID, SAID APPARATUS COMPRISING, A RADIATION SOURCE FOR PROVIDING ILLUMINATION WITHIN AN EXCITATION BAND OF SAID DYE, MEANS FOR DIRECTING SAID ILLUMINATION INTO SAID SPECIMEN, MEANS FOR VIEWING FLUORESCENCE EMISSION PRODUCED FROM SAID DYE RESPONSIVELY TO SAID ILLUMINATION, AND MEANS FOR SPATIALLY FILTERING AT LEAST ONE OF SAID ILLUMINATION AND SAID FLUORESCENCE EMISSION SO AS TO PROVIDE INTENSITY MODULATION OF SAID EMISSION DUE TO BROWNIAN MOTION OF SAID PARTICLES.
 2. Apparatus as defined in claim 1 wherein said means for spatial filtering is disposed in the optical path between said source and said specimen.
 3. Apparatus as defined in claim 1 wherein said means for spatial filtering is disposed in the optical path between said means for viewing, and said specimen.
 4. Apparatus as defined in claim 1 wherein said means for spatial filtering includes one or more light transmissive portions, substantially all of which are dimensioned to be as closely matched to the average dimension of said particles as the wavelengths of said illumination permits.
 5. Apparatus as defined in claim 1 including means for detecting intensity modulation of fluorescence produced by Brownian motion of said particles in combination with said means for spatially filtering.
 6. Apparatus as defined in claim 5 including means for frequency analyzing said intensity modulation.
 7. Apparatus as defined in claim 5 including means for dividing said fluorescence emission into a plurality of beams, said means for detecting including a plurality of wavelength filters each disposed in the path of a respective one of said beams and a corresponding photodetector responsive to the wavelength passed by its corresponding wavelength filter.
 8. Apparatus as defined in claim 7 including means for frequency analyzing the signals produced by said photodetectors.
 9. Apparatus as defined in claim 1 wherein said radiation source comprises a source of broadband radiation and at least one wavelength filter.
 10. Apparatus as defined in claim 1 including means for detecting and measuring scattering of said illumination from said particles substantially simultaneously with viewing of fluorescence emission from said particles.
 11. Process for detection and classification, in a fluid specimen, of submicron dimensioned particles which contain a nucleic acid, which particles are stained by a fluorescent stain specific to said nucleic acid, said process comprising the steps of illuminating said fluid with radiation at an excitation wavelength of said stain observing substantially any fluorescent emission produced from said stain responsively to the excitation illumination; and spatially filtering at least one of the excitation illumination and said fluorescent emission so that the observed fluorescent emission is modulated as a function of the Brownian velocity of the stained particles.
 12. Process as defined in claim 11 including the step of first staining saiD specimen with said fluorescent stain.
 13. Process as defined in claim 11 including the steps of suspending the stained particles in a fluid to form said fluid specimen.
 14. Process as defined in claim 11 wherein said stain is specific to DNA.
 15. Process as defined in claim 11 wherein said stain is specific to RNA.
 16. Process as defined in claim 11 including the steps of staining said particles with both DNA-specific and RNA-specific fluorochromes, and filtering the fluorescence emission at separate wavelengths corresponding to fluorescent wavelengths of stains produced by linkage of said fluorochromes to DNA and RNA, respectively.
 17. Process as defined in claim 11 and further comprising the steps of viewing light scattered from said particles and correlating the observation thereof with the observed spatially filtered fluorescent emissions to determine the ratio of nucleic acid mass to total mass of suspended particles.
 18. Process as defined in claim 17 wherein the fluorescent emissions and scattered light are viewed simultaneously.
 19. Process as defined in claim 11 wherein only said excitation illumination is spatially filtered.
 20. Process as defined in claim 11 wherein only said observed fluorescent emission is spatially filtered.
 21. Process as defined in claim 11 wherein both said excitation illumination and said observed fluorescent emission are spatially filtered.
 22. Process as defined in claim 11 including the step of measuring the frequency spectrum of the modulation of said fluorescent emission.
 23. Process as defined in claim 22 wherein said frequency spectrum is measured at separate fluorescent emission wavelengths respectively characteristic of DNA and RNA.
 24. Apparatus as defined in claim 10 wherein said means for detecting and measuring scattering is sensitive to polarization of the scattered illumination.
 25. Process as defined in claim 17 including the step of measuring polarization of the light scattered from said particles. 